PROJECT SUMMARY The long term goal of this project is to advance our understanding of the cellular and molecular mechanisms that control neural crest development. Neural crest cells are vertebrate-specific, appear early in development, migrate extensively and differentiate into several derivatives, including: craniofacial components (muscle and cartilage amongst others), peripheral nervous system and melanocytes of the skin. Defects in neural crest development and homeostasis result in human pathologies known as neurocristopathies that include cleft lip/palate, Waardenburg syndrome, and melanoma, amongst others. Advancing our understanding of the biology of neural crest cells is fundamental to aid in diagnostic and therapeutic approaches. This project investigates early stages of neural crest formation, and focuses on the contributions made by signaling pathways. This work centers in two models - the avian embryo and a novel model of human neural crest development based on human embryonic stem cells. In the chick we utilize blastula embryos, while in the human stem cell model we address the earliest time points of differentiation. In both models, the role of Wnt and FGF signaling pathways will be scrutinized with particular focus on transcriptional effectors. Aim 1 is directed to elucidate the specific contributions made by distinct FGF-signaling branches during blastula and gastrula stages, and to establish differential responses and transcriptional effectors of the Pea3 sub-family. Aim 2 dissects the contributions made by the dominant canonical and alternative non- canonical Wnt pathways. Here emphasis will be directed to the transcriptional factors Tcf and Lef. Aim 3 will validate and eficcient model of human NC development, and establish the contributions made by these two signaling pathways (FGF, Wnt) to human neural crest development using an efficient and robust model based in human embryonic stem cells. This work will illuminate fundamental principles of neural crest formation in higher vertebrates (birds and mammals), and will invigorate human neural crest studies.